The proposed studies seek to integrate advances in the understanding of the molecular mechanisms controlling insulin secretion and knowledge about tissue specific expression patterns of key islet proteins into an experimental approach to design non-islet cells which have regulated insulin secretion. This proposal will test the hypotheses that most molecules involved in the regulation of insulin secretion are not beta cell-specific and that the specificity for regulation of insulin secretion arises from expression of a small number of mostly beta cell-specific proteins in combination with a "infrastructure" that is common to cells with regulated secretion (neurons and neuroendocrine cells). This application will utilize primary neuroendocrine cells (pituitary and adrenomedullary) which express most, but not all proteins, required for physiologically- regulated insulin secretion (lack GLUT2 glucose transporter, glucokinase (GK), glucagon-like peptide-I (GLP-1) receptor, and glucose-dependent insulin releasing polypeptide (GIP) receptor). In Aim number 1, primary neuroendocrine cells, which have been modified in vitro to secrete human insulin in response to physiologic concentrations of GLP-I, will be transplanted into normal or diabetic rats. The effect of the transplanted cells on glucose homeostasis and their ability to reverse diabetes will be examined. This will examine the ability of non-glucose secretogogues to regulate insulin release from neuroendocrine cells. Aim number 2 investigates whether glucose-regulated insulin secretion can be recreated in neuroendocrine cells. In Aim number 2a, we will examine the function of the ATP-sensitive K+ channel in neuroendocrine cells and its role in hormone secretion by insulin-secreting, neuroendocrine cells. In Aim number 2b and number 2c, which build on the results of Aim number 2a, glucose-stimulated insulin secretion will be reconstituted in neuroendocrine cells by expressing GLUT2 and GK in neuroendocrine cells. Our approach will utilize detailed characterization of hormone secretion in a cell perfusion system, adenovirus-mediated gene transfer, molecular characterization of glucose metabolism and ion channel activity, cell transplantation, and animal studies. The engineered cells will not be subject to immune attack since these will be syngeneic transplants of cells which are not targeted by the autoimmune process of Type I diabetes. The outlined studies expand the horizon beyond engineering cells to simply possess glucose-regulated insulin secretion by developing insulin-secreting cells which are responsive to other physiologic regulators of insulin secretion such as GLP-I or GIP. These studies will allow primary neuroendocrine cells to be isolated from an animal or individual, manipulated in vitro, and then transplanted back into the same animal or individual. The new knowledge generated by this proposal will be integrated with advances in the gene delivery field to develop non-islet cells which secrete insulin in response to physiologic stimuli as therapy for human diabetes.